The long term objective of the proposed research is to understand how genes are switched on and off in response to specific signals. The human interferon-beta gene (IFN-beta) will be used as a model system to approach this problem. The specific aims of the project will be to characterize the structure and function of the multicomponent transcriptional enhancer complex of the IFN-beta gene (the IFN-beta enhanceosome), to investigate the role of chromosomal context and global chromatin structure in the regulation of IFN-beta gene expression, and to characterize a new family of IkappaB kinases. Structural studies will include the application of nuclear magnetic resonance and X-ray crystallography to determine the structure of specific components of the enhanceosome; and single particle visualization of the enhanceosome by atomic force microscopy and electron microscopy. The role of chromosome context and global chromatin structure will be approached by identifying and characterizing long range regulatory sequences within the IFN gene cluster. Comparative genomic sequence information and chromatin accessibility studies will be used to identify putative regulatory sequences. The functional importance of these sequences will then be examined by introducing large regions of the gene cluster on bacterial artificial chromosomes (BACs) into cells in culture. Putative regulatory sequences will be deleted or altered and the consequences on IFN gene expression determined. Finally, with respect to the new family of IkappaB kinases, studies will be carried out to determine their role in the activation of NF-kappaB in different cell types and to identify and clone both upstream and downstream kinases. If successful, these studies will provide significant new insights into the mechanisms of gene regulation and signal transduction, both of which are of fundamental importance in the understanding and treatment of human diseases.